Method for analyzing nucleic acid reactions

Abstract

Disclosed are methods for analyzing reactions involving nucleic acids. The invention utilizes nucleic acids immobilized on a defined substrate in such a manner that the nucleic acid can participate in enzymatic and chemical reactions. These reactions are carried out in the presence of labeled reagents, thereby enabling the progress of the reactions to be analyzed using various techniques, such a fluorescent microscopy. The invention is particularly well suited for investigating transcription phenomena and generating genome-wide maps based upon reactions of individual nucleic acid molecules.

Description

1. INTRODUCTIONThe present invention relates to methods for analyzing nucleic acid reactions in general and transcription reactions in particular. The method utilizes individual nucleic acid molecules immobilized along their length on a planar substrate. Using optical techniques, such as epifluorescent microscopy, the reactions of individual nucleic acid molecules can be studied using the method described herein.The present invention also relates to scalable and massively automatable methods for imaging nucleic acid reactions, either in stop-action fashion or in real time. Bayesian inference estimation methods are utilized to analyze a population of images and to produce data sets of genome-sized scale to be used in the identification of genes, promoter regions, termination regions, or virtually any other phenomena associated with transcription or reverse-transcription of nucleic acid molecules. The method can be used to fabricate maps of transcription events, to correlate these map...

AI Technical Summary

Problems solved by technology

The analysis of nucleic acid molecules at the genome level is an extremely complex endeavor which requires accurate, rapid characterization of large numbers of often very large nucleic acid molecules via high throughput DNA mapping and sequencing.

The construction of physical maps, and ultimately of nucleotide sequences, for eukaryotic chromosomes currently remains laborious and difficult.

However, this method is time-consuming, labor-intensive and expensive, requiring the analysis of four sets of radioactively labeled DNA fragments resolved by gel electrophoresis to determine the DNA sequence.

Current large-scale sequencing is largely the domain of centers where costly and complex support systems are essential for the production efforts.

However, these methods are still dependent on Sanger sequencing reactions and gel electrophoresis to generate ladders and robotic sample handling procedures to deal with the attending numbers of clones and polymerase chain reacting products.

None of the recently developed methods is capable of sequencing individual nucleic acid molecules.

However, extensive high resolution maps of YACs have not been widely generated, due to the high frequency of rearrangement / chimerism among YACs, the low complexity of fingerprints generated by hybridization approaches, and the extensive labor required to overcome these problems.

Limitations of these approaches described above include low throughput, DNA fragmentation (preventing subsequent or simultaneous multimethod analyses), and difficulties in automation.

Despite the potential utilities of these and other approaches, it is increasingly clear that current molecular approaches were developed primarily for characterization of single genes, not entire genomes, and are, therefore, not optimally suited to the analysis of polygenic diseases and complex traits, especially on a population-wide basis (Risch et al., 1996, Science 273:1516-1517).

However, closed systems have limited access to the samples and cannot readily accommodate arrayed samples (Bensimon et al., 1994, Science 265:2096-2098 and Meng et al., 1995, Nature Genet.

USA 88:3233-3237), such approaches were not designed to work with single molecule substrates and could not be relied upon to deposit molecules retaining significant accessibility to enzymatic action.

While single molecule techniques offer the potential advantage of an ordering capability which gel electrophoresis lacks, none of the current single molecule techniques can be used, on a practical level, as high resolution genomic sequencing tools.

Further, while the FISH technique offers the advantage of using only a limited number of immobilized fragments, usually chromosomes, it is not possible to achieve the sizing resolution available with gel electrophoresis.

These techniques, however, are not suited to genome analysis.

First, the steps involved are time consuming and can only be accomplished with a small number of molecules per procedure.

Further, in general, the tethered molecules cannot be stored and used again.

Gridded sample arrays facilitate biochemical manipulations and analyses and are limited only by sample density and available biochemistries.

Conveniently, application of outside forces are completely obviated in the fluid fixation technique, thereby making use of electrical fields, a travelling meniscus or end-tethering of molecules unnecessary.

Obviously, however, deciphering the nucleotide sequence of the human genome is only the first and perhaps smallest step in the process.

The same problem exists in the study of other, less massive genomes, such as those of bacteria and virus.

While the approaches and systems developed for large-scale sequencing have laid the foundation for a broad range of high-throughput systems for molecular analysis, these approaches are not well-suited for studying biochemical mechanisms associated with transcription.

While these approaches analyze the expression levels of thousands of genes simultaneously, they each suffer from insurmountable limitations, such as scalability, speed, and ease of automation.

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